Our Project Goals are twofold: (1) to provide new insight into the catalytic mechanism of cysteine proteases, and (2) to apply this insight toward the rational design of cathepsin B inhibitors for the treatment of cancer and other diseases. Our previous theoretical studies and structural evidence indicate that a unique through-space C(O)NH...S Lewis acid-base interaction plays an essential role in the catalytic mechanism. This interaction delocalizes electronic charge away from the reaction site, thereby stabilizing the transition-state structure and facilitating catalysis. The subject cathepsin-B inhibitors are 1,2-diboronic acid ((OH)2B-CHR-CHR'-B(OH)2] and related compounds, selected as powerful bidentate Lewis-acid ligands that act as transition-state [TS] analogs chelating the enzyme's catalytic thiol group. The substituents R and R' are chosen by inhibitor-enzyme docking simulations maximize the inhibitors' selectivity for cathepsin B. Pursuant to these Project Goals, we propose the following Specific Aims: Specific Aim 1: We will simulate the entire deacylation reaction away of the cysteine proteases papain and cathepsin B. The classical and quantum mechanical calculations include the active-site residues, solvent molecules, and the enzyme environment in order to capture the key reaction components. One key objective is to identify and confirm the transition- state structure for the reactions since it serves as the template for designing the cathepsin-B inhibitors in Specific Aim 2. Specific Aim 2: We will apply our understanding of the catalytic mechanism of cysteine proteases to design and synthesize 1,2 diboronic acids [(OH)2B-CHR-CHR'-B(OH)2] and related compounds as therapeutic inhibitors of cathepsin B for the treatment of cancer and other diseases. These novel inhibitors act as bidentate Lewis acids which chelate the enzyme's catalytic -SH. The substituents R and R' will be selected by docking simulations to maximize the inhibitors' selectivity for cathepsin B. Specific Aim 3: We will systematically correlate calculated and experimental data on these cysteine proteases to critically evaluate alternative catalytic models proposed by others. These include (a) Carey's N...S breakage model, (b)a "field effect" model, and (c) a N-H...S hydrogen-bond model. Of particular interest is their possible relevance to inhibitor design. This project combines computer-aided design, chemical synthesis, and biological evaluation of the subject inhibitors. We have recruited several expert scientists to collaborate without compensation: (i) Dr. Carol Huber, an X-ray crystallographer at the National Research Council of Canada, will perform X-ray crystallographic structural analysis of our inhibitors complexed with both papain and human cathepsin B; (ii) Prof. Lawrence Barton, an experienced boron chemist at UM-St. Louis, will assist in the synthesis and characterization of the diboronic acids; and (iii) Prof. Valerian D'Souza, a bio-organic chemist at UM-St. Louis, will conduct the enzyme assays to evaluate the inhibitory activity of our diboronic acids against papain and cathepsin B. We have recently acquired the X-ray crystallographic coordinates of human liver cathepsin B, courtesy of Prof. Robert Huber, to expedite our inhibitor-design efforts. Copies of letters to confirm these arrangements are included after the Narrative.